Discovery of new natural nisin variants and design, production and characterization of differently engineered lanthipeptides

Antimicrobial resistance is a significant global health concern. Peptide-based therapeutics, known for their selectivity, efficacy, and safety, have gained increased attention due to their novel modes of action and low resistance rates. Ribosomally synthesized and post-translationally modified peptides (RiPPs), the most diverse category of antimicrobial peptides, are promising candidates with slow resistance rates. Nisin, a well-known RiPP, is widely used in various food applications and extensively studied for its genetic organization, biosynthesis, and mode of action. The distinctive biosynthetic pathway and low genetic complexity of lantibiotics make them promising for synthetic biology and bioengineering. Nevertheless, nisin's peptidic nature poses challenges for drug development, such as susceptibility to proteolytic degradation, hindering its potential as a novel antibiotic. In this thesis, we focus on discovering novel nisin variants and engineering them for biomedical applications. Through genome mining, we identified two naturally occurring short nisin variants: cesin from Clostridium estertheticum and rombocin from Romboutsia sedimentorum, featuring 3 and 4 rings in their peptide structures, respectively. Diverse strategies for lanthipeptide engineering were employed, including point mutations, hybrid peptide construction, incorporation of non-canonical amino acids, and post-chemical modifications. Our aim was to overcome nisin's limitations, such as solution stability, protease resistance, and specificity, to enhance its suitability for therapeutic applications. Our results highlight the efficacy of our lanthipeptide engineering methodology in generating novel antimicrobial products, expanding the toolkit for peptide drug improvement and discovery.